Eukaryotic cells are routinely exposed to agents that cause DNA damage and arrest of DNA replication. In response, an as-yet unidentified signal activates highly-conserved signal transduction pathways, termed DNA checkpoints, that halt cell-cycle progression and promote DNA repair. The focus of this proposal is on the role of Replication Protein A (RPA) in this response. RPA is a heterotrimeric ssDNA binding protein that is found in all eukaryotic cells from yeast to humans. RPA's function in DNA replication, repair, and recombination is well understood, but its role in the DNA damage response is unclear. RPA appears to have two roles; it is needed early to fully activate the response, but is also phosphorylated by checkpoint kinases such as Mec1 in yeast. The proposed experiments will address two basic questions: (1) What is the molecular signal that initiates the DNA damage response; and (2) Does phosphorylation of RPA affect its function in DNA repair? Biochemical and genetic approaches in the yeast S. cerevisiae will be used to test the following hypotheses: (1) part of the damage signal consists of RPA bound to ssDNA; and (2) phosphorylation alters RPA's ssDNA binding activity or its interaction with repair and recombination proteins. In Aim 1 the function of damage-dependent RPA phosphorylation will be determined by identifying the sites of modification, mutating them, and testing the mutant alleles for checkpoint function in yeast. Mutant and phosphorylated forms of RPA will be assayed for altered ssDNA binding activities or altered interactions with recombination proteins. In Aim 2 the signal that initiates the DNA damage response will be examined by establishing an in-vitro assay with the Mecl kinase. We will purify Mec1, characterize its enzymatic activity, and search for activators. Various DNA substrates, in the absence and presence of RPA, will be tested for activator function. The ability of mutant or phosphorylated RPA to stimulate Mec1- and Rad53- kinases will also be assayed. In Aim 3 potential regulatory domains of Mec1 will be identified by performing a structure/function analysis. The hypothesis that Mecl regulates separate DNA repair and checkpoint pathways will be tested genetically by searching for MEC1 alleles that affect DNA repair uniquely. These studies will provide insight into the nature of the DNA damage signal and the regulation of checkpoint kinases. The results are expected to have broad implications for the mechanism of DNA repair in human cells.